SNARE-dependent glutamate release in megakaryocytes Catherine J. Thompson, Tatjana Schilling, Martin R. Howard, Paul G. Genever  Experimental Hematology  Volume 38, Issue 6, Pages 504-515 (June 2010) DOI: 10.1016/j.exphem.2010.03.011 Copyright © 2010 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 1 Expression of soluble N-ethyl maleimide-sensitive factor attachment protein receptor (SNARE) complex and accessory genes by megakaryocytes. Reverse transcription polymerase chain reaction was used to determine messenger RNA expression of SNARE complex and accessory proteins in MEG-01 cells (left panel), primary murine megakaryocytes (MKs, middle panel) and primary human MKs (right panel). For MEG-01 cells, expression in the undifferentiated (−phorbol myristate acetate [PMA]), as well as differentiated (+PMA) state is shown. Human fetal forebrain or mouse whole brain was used as a positive control. Negative controls (no reverse transcriptase [RT] and water) were performed in parallel. Experimental Hematology 2010 38, 504-515DOI: (10.1016/j.exphem.2010.03.011) Copyright © 2010 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 2 Western blot analysis of soluble N-ethyl maleimide-sensitive factor attachment protein receptor (SNARE) complex proteins in megakaryocytes. Expression of SNARE complex proteins SNAP-23, Syntaxin 4, and vesicle-associated membrane protein (VAMP) was determined by Western blot analysis in MEG-01 cells, in the undifferentiated (−phorbol myristate acetate [PMA]), as well as differentiated (+PMA) state, and in primary human megakaryocytes (MK). Human brain (HB) lysate was used as a positive control, detection of glyceraldehyde phosphate dehydrogenase (GAPDH) was performed as loading control. Experimental Hematology 2010 38, 504-515DOI: (10.1016/j.exphem.2010.03.011) Copyright © 2010 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 3 Immunolocalization of soluble N-ethyl maleimide-sensitive factor attachment protein receptor (SNARE) complex and accessory proteins in megakaryocytes. (A) Immunofluorescent localization of SNARE complex and accessory proteins (green) was determined by confocal laser scanning microscopy in MEG-01 cells in the undifferentiated (−phorbol myristate acetate [PMA]) as well as differentiated (+PMA) state, nuclei were stained with 4′,6-diamidino-2-phenylindole (blue); the bottom row shows negative controls for vesicle-associated membrane protein (VAMP) and Syntaxin (mouse immunoglobulin G) and for Synaptotagmin, SNAP-23, vesicular glutamate transporter (VGLUT) 1, and VGLUT2 (rabbit immunoglobulin G) immunostainings, as well as the overexpression of enhanced green fluorescent protein (EGFP)-VGLUT1 in undifferentiated (−PMA) MEG-01 cells and empty vector control (EGFP con); arrows indicate elevated accumulation of VGLUT1 and VGLUT2 at the cell periphery in differentiated MEG-01 cells as well as dense distribution of VGLUT1 in specific regions near the plasma membrane of VGLUT1-transfected cells; bar = 20 μm. (B) Brown staining reports expression of SNARE complex proteins (Syntaxin 4 and SNAP-23) and VGLUT1/2, respectively, in rat bone marrow. Detection of CD61 was used to confirm megakaryocyte identity. Nuclei were counterstained with hematoxylin (blue); bar = 30 μm. Experimental Hematology 2010 38, 504-515DOI: (10.1016/j.exphem.2010.03.011) Copyright © 2010 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 4 Identification of acidic, recycling vesicles in megakaryocytes. (A) After acridine orange staining, acidic vesicles were observed in the MEG-01 cells (red) in the periphery of the cells. Green fluorescence reports soluble dye within the cytosol. (B) Acidic vesicles were also observed in primary human megakaryocytes (MKs), where additionally to their localization at the periphery of the cells (red), they colocalized with soluble dye in the cytosol of the cell (yellow); bars = 20 μm. (C) Vesicular recycling in primary murine MKs was detected by incorporation of the FM1-43 dye into vesicle membranes during endocytosis, which leads to an increase of green fluorescence within the cells. Following FM1-43 removal, the cells destained within 60 minutes reporting exocytosis of these vesicles. Experimental Hematology 2010 38, 504-515DOI: (10.1016/j.exphem.2010.03.011) Copyright © 2010 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 5 Glutamate release in megakaryocytes. (A) After 72 hours of cultivation, an enzyme-linked fluorimetric assay was used to detect glutamate release from MEG-01 cells in the undifferentiated (−phorbol myristate acetate [PMA]) as well as differentiated (+PMA) state and from primary murine and human megakaryocytes (MKs) (n = 3, mean + standard deviation; ∗∗∗p < 0.001). (B) Western blot analysis (evaluated by densitometry normalized to the housekeeping protein glyceraldehyde phosphate dehydrogenase [GAPDH]) demonstrated that treatment with 100 nM tetanus toxin decreased vesicle-associated membrane protein (VAMP) protein levels in MEG-01 cells in the undifferentiated (−PMA; 45% decrease compared to undifferentiated control) as well as differentiated (+PMA; 94% decrease compared to differentiated control) state, which inhibits formation of the SNARE complex. (C) Using the enzyme-linked fluorimetric assay with MEG-01 cells overexpressing the tetanus toxin light chain (TeTxLC) demonstrated a 30% reduced glutamate release as a result of the inhibition of the SNARE complex due to reduction of VAMP compared to empty vector control (pcDNA3.1/V5-His B; n = 3, mean + standard deviation; ∗∗∗p < 0.001). (D) Two different transfection vectors (pcDNA3.1/V5-HIS B and pEGFP-N2) were used to introduce VGLUT1 overexpression into MEG-01 cells. Glutamate released from VGLUT-overexpressing cells, as well as intracellular glutamate was detected by an enzyme-linked fluorimetric assay. Compared to empty vector controls (pcDNA3.1/V5-His B and pEGFP-N2, respectively), glutamate release was 2.26- and 1.69-fold increased and intracellular glutamate was 2.29- and 1.69-fold elevated in pcDNA3.1/V-His B hVGLUT1- and pEGFP-N2 VGLUT1-transfected cells, respectively (n = 3, mean + SD; ∗∗∗p < 0.001). Experimental Hematology 2010 38, 504-515DOI: (10.1016/j.exphem.2010.03.011) Copyright © 2010 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 5 Glutamate release in megakaryocytes. (A) After 72 hours of cultivation, an enzyme-linked fluorimetric assay was used to detect glutamate release from MEG-01 cells in the undifferentiated (−phorbol myristate acetate [PMA]) as well as differentiated (+PMA) state and from primary murine and human megakaryocytes (MKs) (n = 3, mean + standard deviation; ∗∗∗p < 0.001). (B) Western blot analysis (evaluated by densitometry normalized to the housekeeping protein glyceraldehyde phosphate dehydrogenase [GAPDH]) demonstrated that treatment with 100 nM tetanus toxin decreased vesicle-associated membrane protein (VAMP) protein levels in MEG-01 cells in the undifferentiated (−PMA; 45% decrease compared to undifferentiated control) as well as differentiated (+PMA; 94% decrease compared to differentiated control) state, which inhibits formation of the SNARE complex. (C) Using the enzyme-linked fluorimetric assay with MEG-01 cells overexpressing the tetanus toxin light chain (TeTxLC) demonstrated a 30% reduced glutamate release as a result of the inhibition of the SNARE complex due to reduction of VAMP compared to empty vector control (pcDNA3.1/V5-His B; n = 3, mean + standard deviation; ∗∗∗p < 0.001). (D) Two different transfection vectors (pcDNA3.1/V5-HIS B and pEGFP-N2) were used to introduce VGLUT1 overexpression into MEG-01 cells. Glutamate released from VGLUT-overexpressing cells, as well as intracellular glutamate was detected by an enzyme-linked fluorimetric assay. Compared to empty vector controls (pcDNA3.1/V5-His B and pEGFP-N2, respectively), glutamate release was 2.26- and 1.69-fold increased and intracellular glutamate was 2.29- and 1.69-fold elevated in pcDNA3.1/V-His B hVGLUT1- and pEGFP-N2 VGLUT1-transfected cells, respectively (n = 3, mean + SD; ∗∗∗p < 0.001). Experimental Hematology 2010 38, 504-515DOI: (10.1016/j.exphem.2010.03.011) Copyright © 2010 ISEH - Society for Hematology and Stem Cells Terms and Conditions